Study On Purification And Chemical Modification Of Trypsins From Chinese Sucker (Myxocyprinus Asiaticus)

Trypsin (EC 3.4.21.4) is a member of a large family of serine proteases,which is detected in the pancreas , pyloric ceca and intestine of animals.They work as digestive enzymes in the wertebrate.Trypsin synthesis in the pancreas and secreted into digestive tract in the form of zymogen with non-activity.In the digestive tract it changed into the active form by removing part of the peptide chain of zymogen, and exercised the digestive function.Therefore, the chemical modification of trypsin was researched. In this study, the trypsin was purified from Chinese sucker (Myxocyprinus asiaticus),and then the trypsin was modified with activated dextran (T-70). By the experiments, the optimum conditions for chemical modification were selected, and the main properties of the trypsin before and after the modification were also studied by comparison.1. The purification of trypsin from Chinese sucker (Myxocyprinus asiaticus)The hepatopancreas of Chinese sucker were separated and washed with double distilled water, and then homogenized with 50 mM (pH 8.0) Tris–HCl containing Ca2+ at a ratio of 1:5 (w/v).The mixture was stored overnight at 4°C then centrifuged .The supernatant was separated which was considered the crude enzyme extract. It was subjected to ammonium sulphate fractionation in the 20-80% saturation range and the precipitate was collected by centrifugation. The precipitate was dissolved in Tris–HCl buffer and dialyzed 24 h at 4℃and continuously changed the buffer. Using HiTrap Benzamidine FF(high sub) affinity equied column (5ml) ,the 50mM Gly-HCl buffer (pH 3.0)worked as eluent. The protein peaks were collected and detected their activity . In the time accommodated its pH value to 9.0. Then the fractions were loaded into a DEAE-Sepharose TM Fast Flow column (Amersham Pharmacia Biotech).And the column was eluted with a linear gradient ranging from 0 to 0.5 M NaCl. The enzyme protein content was collected after each purified step .The result shows that the final preparations from Chinese sucker were about 185.3-fold, with the recovery of 8% from the crude trypsin.2. Modification of trypsin from Chinese sucker (Myxocyprinus asiaticus)The trypsin from Chinese sucker was modified by NaIO4 oxidized dextran, the modification rate was affected by the reaction conditions. The optimization of the reaction condition can enhance the modification rate and the reservation of enzyme activity. And the optimum reaction conditions were as follows: the ration of NaIO4 and dextran was 1:1, the ration of oxidized dextran and trypsin was 5:1, and the reaction time was 4h. Under these conditions, the modification rate was 87%, the reservation of enzyme activity was 53%.3. Comparative study on the characterization of trypsin before and after enzyme modificationSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to determined the changes of molecular weight. The purified trypsin from Chinese sucker show a single band and the molecular weight was estimated as 26KDa, and in the molecular weight of 66 KDa, the modified enzyme shows a main band following the diffusion of small molecular weight band.The Michaelis constant (Km) of trypsins and the modification enzymes determined to be 0.074mM,0.21mM respectively by using BAPNA (Nα-benzoyl-DL–arginine -p - nitroanilide) method.For the hydrolysis of TAME(N-p-tosyl-L-arginine methyl ester)hydrochloride ,the optimum temperature for trypsins were 60℃and the optimum pH of trypsins were 8.5. At pH6.0-10.0,trypsin was stable. Under the same conditions, the modification enzymes kept the same the optimum temperature and pH. The pH stability of the trypsin before and after modification kept the same trend roughly. Enzymatic reaction kinetics shows that the modification of the catalytic efficiency is lower than the original enzyme, mainly due to the substrate affinity of the enzyme decreasing.